Methods are known for detecting specific nucleic acids or analytes in a sample with high specificity and sensitivity. Such methods generally require first amplifying nucleic acid sequence based on the presence of a specific target sequence or analyte. Following amplification, the amplified sequences are detected and quantified. Conventional detection systems for nucleic acids include detection of fluorescent labels, fluorescent enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels.
One disadvantage of detection methods presently widely in use is the need to separate labeled starting materials from a final labeled product or by-product. Such separations generally require gel electrophoresis or immobilization of a target sequence onto a membrane for detection. Moreover, there are often numerous reagents and/or incubation steps required for detection.
It has been known that DNA and RNA polymerases are able to recognize and utilize nucleosides with a modification at or in place of the gamma position of the triphosphate moiety. It is further known that the ability of various polymerases to recognize and utilize gamma-modified nucleotide triphosphates (NTP's) appears to vary depending on the moiety attached to the gamma phosphate. In general, RNA polymerases are more promiscuous than DNA polymerases.
A colorimetric assay for monitoring RNA synthesis from RNA polymerases in presence of a gamma-phosphate modified nucleotide has been previously reported. In this prior report, RNA polymerase reactions were performed in the presence of a gamma-modified, alkaline phosphatase resistant nucleotide triphosphate which was modified at its gamma-phosphate with a dinitrophenyl group. When RNA polymerase reactions were performed in the presence of this gamma-modified NTP as the sole nucleotide triphosphate and a homopolymeric template, it was found that RNA polymerase could recognize and utilize the modified NTP. Moreover, when the polymerase reactions were performed in the presence of an alkaline phosphatase, which digested the p-nitrophenyl pyrophosphate aldo-product of phosphoryl transfer to the chromogenic p-nitrophenylate, an increase in absorbence was reported. A disadvantage of this detection method is that the real-time colorimetric assay, performed in the presence of an alkaline phosphatase, only works with a homopolymeric template.
It would, therefore, be of benefit to provide a method for detecting RNA in the presence of a heteropolymeric template, which method would not be restricted to using a single terminal-phosphate modified nucleotide as the sole nucleotide that is substantially non-reactive to alkaline phosphatase. This would allow for a single-tube assay for real-time monitoring of RNA synthesis using hetero-polymeric templates.
Generally, the fluorescent dyes in the assays are quenched by a molecule placed in close proximity to them in the labeled entity, and the detectable signal is produce when the structure is altered and the quencher is either removed, moved away from the dye, or otherwise rendered inactive. At that point a detectable signal is produced. However, since quenching is not absolute, the dynamic range of such assays is limited.
It would further be of benefit to provide for similar assays for RNA wherein the identity of the label on the terminal-phosphate is varied to allow for better recognition and utilization by RNA polymerase. Furthermore, it is desired that the label on the terminal-phosphate could be varied so as to allow for chemiluminescent and fluorescent detection, or reduction potential, as well as for improved calorimetric detection, wherein only simple and routine instrumentation would be required for detection, and increased dynamic range.
Given that DNA polymerases are known in the art to be less promiscuous than RNA polymerases regarding recognition and utilization of terminally-modified nucleotides, wherein the identity of the moiety at the terminal position can largely affect the DNA polymerase's specificity toward the nucleotide, it would be highly desired to provide for a non-radioactive method for detecting DNA by monitoring DNA polymerase activity. Furthermore, it would be desired that the synthesis and detection of DNA could be accomplished in a single-tube assay for real-time monitoring and that the label at the terminal-phosphate of nucleotide substrates could encompass chemiluminescent, fluorescent, and calorimetric detection, as well as analysis by mass or reduction potential.